The enzyme 11-hydroxylase (CYP27b1; the rate limiting enzyme converting 25-hydroxyvitamin D3 to the most active form of vitamin D3, 1,25-dihydroxyvitamin D3) and the vitamin D receptor (VDR), a nuclear hormone receptor super-family member, are distributed throughout many organ systems and have established roles in the regulation of bone health, the immune system, growth, differentiation and development. Several recent clinical and laboratory studies documenting either subfertility or frank infertility in vitamin D3 deficient states suggest that vitamin D3 may be essential for normal reproductive physiology and reproductive success. The mechanisms by which vitamin D3 regulates the hypothalamic-pituitary-gonadal axis and how vitamin D3 deficiency adversely affects female fertility and reproduction are not understood. Phenotypes in VDR and CYP27b1 knockout mice include hypogonadism, arrested ovarian follicular development and hypoplastic uteri. Uterine responsive to exogenous steroids is intact in CYP27b1 and VDR knockout mice, thus suggesting that vitamin D3 deficiency impairs one or more components of the hypothalamic-pituitary-ovarian axis. The goal of this exploratory project is to test the hypothesis that vitamin D3 deficiency compromises female reproductive success by adversely affecting the neuroendocrine (hypothalamic-pituitary) component of the reproductive axis. The proposed experiments will use CYP27b1 knockout mice, deficient in active 1,25-dihydroxyvitamin D3, as a model to elucidate the mechanisms through which peripubertal and adult vitamin D3 regulates the pubertal transition and the female reproductive system. Specific Aim 1 determines if 1,25-(OH)2D3 is required for normal ovarian physiology. We will determine if 1,25-(OH)2D3 deficiency disrupts follicular development, ovarian steroid production, or renders ovarian follicles less responsive to gonadotropins. Specific Aim 2 determines if 1,25-(OH)2D3 deficiency disrupts hypothalamic-pituitary axis function. Our preliminary data show that gonadotropins stimulate follicular development and ovulation, but that puberty and the first estrus are delayed in vitamin D3 deficient CYP27b1 null female mice and that null mice have altered estrous cycles. These data suggest that vitamin D3 deficiency adversely affects hypothalamic- pituitary physiology. Therefore, we will focus on neuroendocrine function and systematically analyze how vitamin D3 deficiency disrupts the function of gonadotropin releasing hormone (GnRH) neurons and of pituitary gonadotrophs and if the adverse effects of vitamin D3 deficiency is reversible. Vitamin D insufficiency and deficiency have reached near epidemic levels worldwide, with peripubertal children and reproductive aged women being disproportionately affected. Completion of these specific aims will provide critical information regarding the regulatory role of vitamin D3 in female reproduction and may provide insight into the mechanisms underlying reduced reproductive success in vitamin D3 deficient women. PUBLIC HEALTH RELEVANCE: Vitamin D3 deficiency and insufficiency are significant public health problems, affecting as much as 36% of the United States population. Importantly, vitamin D3 deficiency disproportionately affects peripubertal children, reproductive aged women, especially poor women and women of color, thus having the potential to adversely affect reproductive function and pregnancy outcomes. The proposed experiments will provide insight into the mechanisms through which vitamin D3 regulates female reproduction and fertility with a focus on the impact of vitamin D3 deficiency on the integrity of hypothalamic-pituitary-gonadal axis. This project may provide a foundation for the institution of screening algorithms for at risk populations and the development of a relatively safe, inexpensive, biologically plausible yet simple medical intervention intended to eliminate disparities, to improve public health outcomes, and to reduce the burden of health care costs.